Method of increasing the function of an aav vector

Abstract

A method of correcting singletons in a selected AAV sequence in order to increasing the packaging yield, transduction efficiency, and/or gene transfer efficiency of the selected AAV is provided. This method involves altering one or more singletons in the parental AAV capsid to conform the singleton to the amino acid in the corresponding position(s) of the aligned functional AAV capsid sequences.

1 .- 8 . (canceled) 
     
     
         9 . A recombinant adeno-associated virus (AAV) comprising an AAV capsid and a minigene having AAV inverted terminal repeats and a heterologous gene operably linked to regulatory sequences which direct expression of the heterologous gene in a host cell, wherein the AAV capsid comprises AAV vp1 proteins, AAV vp2 proteins, and AAV vp3 proteins, wherein the AAV vp3 proteins have i) the sequence of amino acids 204 to 738 of SEQ ID NO: 4 (AAVrh46), or ii) an amino acid sequence having at least 95% identity to the full length of amino acids 204 to 738 of SEQ ID NO:4, wherein the amino acid residue corresponding to position 665 in SEQ ID NO: 4 is N when aligned along the full length of amino acids 204 to 738 of SEQ ID NO: 4. 
     
     
         10 . The recombinant AAV according to  claim 9 , wherein the AAV vp3 capsid proteins are encoded by nucleotides 610 to 2217 of the nucleic acid sequence of SEQ ID NO: 22. 
     
     
         11 . The recombinant AAV according to  claim 9 , wherein the AAV vp3 capsid protein have an amino acid sequence which is at least 97% identical to the full length of amino acids 204 to 738 of SEQ ID NO: 4, wherein the amino acid residue corresponding to position 665 is N when aligned along the full length of amino acids 204 to 738 of SEQ ID NO: 4. 
     
     
         12 . The recombinant AAV according to  claim 9 , wherein the AAV inverted terminal repeats are from a different AAV than the AAV supplying the capsid proteins. 
     
     
         13 . A composition comprising the recombinant AAV according to  claim 9  and a physiologically compatible carrier. 
     
     
         14 . The recombinant AAV according to  claim 9 , wherein the heterologous gene encodes an ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, carbamoyl phosphate synthetase I, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, cystathione beta-synthase, branched chain ketoacid decarboxylase, isovaleryl-coA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase (GCDH), betaglucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, β-glucuronidase (GUSB), glycine decarboxylase, a low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, very low density lipoprotein (VLDL) receptor, scavenger receptor, glucocorticoid receptor, estrogen receptor, Vitamin D receptor, nuclear receptor, cystic fibrosis transmembrane regulator (CFTR) sequence, Factor IX or variants thereof, Factor VIII or variants thereof, a dystrophin gene product, or an immunoglobulin. 
     
     
         15 . The recombinant AAV according to  claim 14 , wherein the dystrophin gene product is a mini-dystrophin or micro-dystrophin. 
     
     
         16 . (canceled) 
     
     
         17 . The recombinant AAV according to claim  1 , wherein the AAV vp3 capsid proteins have an amino acid sequence which is 100% identical to the full length of amino acids 204 to 738 of SEQ ID NO: 4.